Animal Development and Differentiation Research Unit, Division of Animal Sciences, National Institute of Agrobiological Sciences (NIAS), Tsukuba, Ibaraki, Japan.
Biol Reprod. 2013 Aug 8;89(2):28. doi: 10.1095/biolreprod.112.106641. Print 2013 Aug.
Bovine embryonic stem (ES) cells have the potential to provide significant benefits in a range of agricultural and biomedical applications. Here, we employed a combination of conventional methods using glycogen synthase kinase 3 and mitogen-activated protein kinase inhibitors to establish ES cell lines from in vitro fertilization (IVF) and somatic cell nuclear transfer (SCNT) bovine embryos. Five male cell lines were established from IVF embryos, and two female and three male cell lines from SCNT blastocysts; we named these lines bovine ES cell-like cells (bESLCs). The lines exhibited dome-shaped colonies, stained positively for alkaline phosphatase, and expressed pluripotent stem cell markers such as POU5F1, SOX2, and SSEA-1. The expression levels of these markers, especially for NANOG, varied among the cell lines. A DNA methylation assay showed the POU5F1 promoter region was hypomethylated compared to fibroblast cells. An in vitro differentiation assay showed that endoderm and ectoderm marker genes, but not mesoderm markers, were upregulated in differentiating bESLCs. To examine bESLCs in later embryonic stages, we created 22 chimeric blastocysts with a male bESLC line carrying a GFP marker gene and transferred these to a recipient cow. Four chimeric embryos were subsequently retrieved on Day 13 and retransferred to two recipient cows. One living fetus was obtained at Day 62. GFP signals were not identified in fetal cells by fluorescence microscopy; however, genomic PCR analysis detected the GFP gene in major organs. Clusters of GFP-positive cells were observed in amniotic membranes, suggesting that bESLCs can be categorized as a novel type of ICM-derived cells that can potentially differentiate into epiblast and hypoblast lineages.
牛胚胎干细胞（ES 细胞）在农业和生物医学应用领域具有广泛的应用潜力。本研究采用常规方法，结合糖原合成酶激酶 3 和丝裂原活化蛋白激酶抑制剂，从体外受精（IVF）和体细胞核移植（SCNT）牛胚胎中建立 ES 细胞系。我们从 IVF 胚胎中建立了 5 个雄性细胞系，从 SCNT 囊胚中建立了 2 个雌性和 3 个雄性细胞系；我们将这些细胞系命名为牛类胚胎干细胞（bESLC）。这些细胞系表现出穹顶形集落，碱性磷酸酶染色阳性，并表达多能干细胞标志物，如 POU5F1、SOX2 和 SSEA-1。这些标志物的表达水平，尤其是 NANOG，在不同的细胞系中存在差异。DNA 甲基化分析显示 POU5F1 启动子区域与成纤维细胞相比呈低甲基化状态。体外分化试验表明，bESLC 中内胚层和外胚层标志物基因上调，但中胚层标志物基因没有上调。为了研究 bESLC 在胚胎晚期的情况，我们构建了 22 个携带 GFP 标记基因的雄性 bESLC 系的嵌合体囊胚，并将其移植到受体牛中。随后在第 13 天回收了 4 个嵌合体胚胎，并将其重新移植到 2 头受体牛中。第 62 天获得了一头活胎。荧光显微镜未检测到胎儿细胞中的 GFP 信号，但基因组 PCR 分析检测到主要器官中的 GFP 基因。在羊膜中观察到 GFP 阳性细胞簇，提示 bESLC 可归类为一种新型的 ICM 衍生细胞，可能分化为内细胞团和滋养外胚层谱系。
